Exosomes from uterine fluid promote capacitation of human sperm

Ethical approval

The study was approved by the Ethical Committee of the First Affiliated Hospital of Kunming Medical University, China, and all of the participants provided written informed consent. This study was designed and conducted according to the Declaration of Helsinki.

Sperm preparation

Semen was collected by masturbation from healthy donors (N = 3, age 25–35 years) who had been abstinent for 2–7 days. The semen parameters of donors were normal according to the World Health Organization (WHO) guidelines (Björndahl & Kirkman Brown, 2022). Semen was liquefied for 30 min at room temperature, and spermatozoa were isolated from the semen by discontinuous Percoll gradient centrifugation. Sperm were resuspended in Biggers-Whitten-Whittingham (BWW) medium (Solarbio, Beijing, China) and maintained at 37 °C in an incubator with 5% CO₂ in compressed air and high humidity. In the experiments on sperm capacitation, we used a non-capacitating BWW medium (NC-BWW, Hepenbio, China), which did not contain human serum albumin or HCO₃⁻.

Collection and processing of uterine flushes

Ten female volunteers with proven fertility were recruited in this study. Inclusion criteria were: (1) 25–45 years of age, (2) proven fertility, (3) no HPV infection, and (4) no cervical or uterine inflammation. The uterine flush fluid was collected at the Gynecology Department of the First Affiliated Hospital of Kunming Medical University. Briefly, the uterus was flushed with a disposable catheter filled with 40–50 ml of normal saline before hysteroscopic surgery. Samples were immediately collected from the catheter, and the supernatant was filtered through a 0.22-µm filter to remove cells and cellular debris. The supernatant was then transferred to an Amicon Ultra-15 10 K centrifuge filter (Merck Millipore, Rahway, NJ, USA), centrifuged at 5,000 g for 10 min at 4 °C to concentrate it to 2 ml, and stored at −20 °C.

EV isolation

EVs were isolated using ExoQuick TC™ (SBI, CA, USA) according to the manufacturer’s instructions (Alvarez, 2014). Briefly, the supernatant was thoroughly mixed with ExoQuick TC™ at a volume/volume ratio of 2:1, and the mixture was maintained at 4 °C for 12–16 h, followed by centrifugation of 16,000 g at 4 °C for one hour. The supernatant was then discarded. The resulting EV pellet was resuspended in 100 µl of sterile PBS and preserved at −80 °C. EVs from 10 donors were pooled and then incubated with sperm from three different donors.

Transmission electron microscopic analysis of EVs

The quality of isolated EVs was assessed by transmission electron microscopy (TEM) as described previously (Yang et al., 2019).

Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) was performed using a ZeTaView Particle Metrix instrument (Szatanek et al., 2017). Briefly, the samples were first diluted with PBS to achieve 20–120 nanoparticles per frame and/or 10⁷–10⁹ nanoparticles/ml. Three 30-second videos were then recorded at 30 frames per second for each sample. The concentration, mean size and SEM of the nanoparticles in each sample were calculated.

Western blot analysis

EVs were extracted and subjected to protein quantification using a BCA kit and stored at −80 °C. Samples selected for electrophoresis were prepared in SDS-PAGE sample loading buffer and heated for 5 min at 100 °C. Ten micrograms of protein were loaded per lane on 10% polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Blots were then blocked for 2 h at room temperature and incubated with anti-CD63 (1:500, 30–55 kDa; 25682-1-AP, Proteintech, Chigago, IL, USA) and anti-CD9 (1:20,000, 23–30 kDa; 20597-1-AP, Proteintech, Chicago, IL, USA) primary antibodies overnight at 4 °C. Non-specific binding of antibody was removed by washing three times with TBST for 10 min each before incubation with goat anti-rabbit IgG H&L (HRP, 1:10,000) for one hour at 4 °C. The membrane was again washed three times with TBST for 10 min chemiluminescence was detected using an ECL kit (Campoy et al., 2016; Kowal et al., 2017).

Sperm motility

Sperm were incubated with EVs at 37 °C for 2 h and 4 h. Curvilinear velocity (VCL) and percentage of motile sperm were determined using a computer-assisted sperm analysis system (Sperm Class Analyzer; Microptic, Barcelona, Spain).

Measurement of the acrosome reaction

The sperm were equally divided into three groups and incubated with 20 µl of PBS, 20 µl of EVs, or 10 µM calcium ionophore A23187 (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C, respectively. The sperm from all of the groups were fixed in 4% paraformaldehyde for 25 min at room temperature and then washed twice with PBS (Xu et al., 2021).

The fixed sperm were stained with 0.22% Kaumas Brilliant Blue G250 (Sigma Aldrich, St. Louis, MO, USA) for 5 min to visualize sperm heads. After rinsing with distilled water, the sperm were allowed to dry and were fixed with 90% glycerol. The acrosome reaction and morphology were observed and photographed under a light microscope at 400x magnification. We counted 200 cells from each sample (Fig. 1) (Murdica et al., 2019).

Measurement of ROS

Sperm were incubated with EVs for 2 h at 37 °C, centrifuged at 1,400 r/min for 8 min, and the supernatant aspirated. After that, 2′, 7 Dichlorofluorescein diacetate (DCFH-DA, MedChemExpress, Monmouth Junction, NJ, USA) was added and incubated with sperm for 20 min at 37 °C. Sperm were centrifuged twice to remove DCFH-DA, and resuspended in 100 µl of PBS solution and incubated at room temperature in the dark for 20 to 30 min. Fluorescence intensity was determined using flow cytometry (ACEA Biosciences, San Diego, CA, USA).

Statistical analysis

Sperm from three donors were used to conduct experiments, and experiments with each donor’s sperm were replicated three times. We adopted one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test to determine the differences among groups using SPSS 22.0 software. p < 0.05 was considered to be statistically significant.

Characteristics of EVs in uterine flushes

TEM analysis confirmed the presence of EVs in all 10 of the uterine douche samples collected in this study. As shown in representative electron microscopic images (Figs. 2A, 2B), the fraction isolated from the uterine douche showed cup-shaped vesicles of different sizes. All of the vesicles showed a honeycomb-like double-membrane structure with diameters ranging from 30 nm to 200 nm, a typical feature of EVs (Franchi et al., 2016).

Next, further characterization of EVs using NTA showed that all of the isolated vesicles contained similar numbers of vesicles in the expected size range (30–200 nm) (Figs. 2C, 2D) (Szatanek et al., 2017), and that EVs from uterine fluid showed characteristics similar to EVs from EECs cultured in vitro. We detected CD9 and CD63 in EVs from different donors by western blot analysis (Fig. 2E) (Campoy et al., 2016).

Effect of EVs on the motility of human sperm

Sperm incubated with EVs for 2 h were measured using an SQA-V automated semen analyzer, and the data were analyzed using a computer-assisted semen analysis system. We herein considered the percentage of sperm with forward and non-forward motion as an indicator of sperm motility. Our results revealed that sperm motility did not differ at 2 h, but was significantly different at 4 h (Fig. 3).

Effect of EVs on sperm capacitation

It has been demonstrated that uterine fluid promotes sperm capacitation and the acrosome reaction, and we herein assessed sperm capacitation by the level of acrosome reaction. After staining with Kaumas Brilliant Blue and observation by light microscopy, sperm heads with an intact acrosome stained blue, and the regions of the sperm heads after the reaction remained unstained (Lu et al., 2002). We observed acrosome reactions in every group (Fig. 4).

The percentage of unstained sperm acrosomes in group A was significantly lower than in groups B and C, showing that the percentage of acrosome-reacted sperm increased significantly after incubation with EVs or A23187 (Fig. 5). These data suggest that uterine fluid EVs modulate the physiological state of sperm and increase their energetic capacity.

EVs increase levels of ROS in human sperm

The ROS content of the sperm in each group was quantified by measuring the fluorescence intensity using flow cytometry. DCFH-DA is a cell-permeable dye that is hydrolyzed intracellularly to DCFH, and oxidation of DCFH by the action of intracellular ROS transforms the molecule into its highly fluorescent form, DCF (Rajneesh Pathak et al., 2017). When we applied DCFH-DA to detect cytosolic ROS production in sperm, we observed that the fluorescence intensity increased significantly after sperm were incubated with EVs from uterine flushes, indicating an increase in ROS. This result suggests that EVs increased the ROS levels of sperm (Fig. 6).